Use of chemical compounds that can inhibit the toxic activity of sphingomyelinase d from venoms of loxosceles spiders and pharmaceutical composition comprising said compounds

ABSTRACT

The present invention relates preferably to the use of 4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenesulphonamide and 4-methyl-3-oxo-2-(3-pyridylmethylene) benzo[3,4-b]furan-6-yl-4-chlorobenzenesulphonate (compounds 5 and 6, respectively), which are compounds that can inhibit the toxic activity of sphingomyelinase D from  Loxosceles  venom, controlling the development of cutaneous and systemic loxoscelism; reducing haemolysis; inhibiting the formation of skin lesions; inhibiting skin necrosis; inhibiting intracellular signaling pathways and the production of reactive oxygen species. In addition to the therapeutic potential thereof, said inhibitors can be used to study the activity of sphingomyelinases and phospholipases D. The present invention also relates to a pharmaceutical composition for treating loxoscelism, reducing haemolysis, inhibiting the formation of skin lesions, inhibiting skin necrosis, inhibiting intracellular signaling pathways and the production of reactive oxygen species, comprising said compounds and a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

The present invention relates to the use of chemical compounds capableof inhibiting the toxicity of sphingomyelinase D of the venom of spiderLoxosceles. More specifically, the instant invention relates to the useof benzenesulfonamides and benzenesuphonates on toxicity inhibition ofsphingomyelinase D of the venom of spider Loxosceles.

BACKGROUND OF THE INVENTION

Loxoscelism (accident involving brown recluse spider) has been describedin several continents. It corresponds to the most severe form ofaraneism in Brazil. The majority of accidents notified occur in theSouth Region of the country, particularly in Paraná and Santa Catarina.The accident occurs more commonly with adults, with a small predominancein women, occurring at home. A centripetal distribution of insect bitesis observed, which affect more the thigh, the upper body or arm.

The most important component of Loxosceles venom is sphingomyelinase D,that acts on the extracellular matrix and through the activation of thecomplement system and action on endothelial cells, epithelial cells,leukocytes and platelets, which causes the release of inflammatorymediators, obstruction of small vessels at the site of venom inoculationand consequent tissue damage. Likewise, hemolysis has been attributed tothe action of sphingomyelinase-D on endogenous metalloproteinases. Onceactivated, they act on the membrane proteins of red blood cells, makingthem susceptible to complement action.

The Brazilian patent application PI 0514809-0 A, filed on Aug. 29, 2005,on behalf of National Autonomous University of Mexico) and LaboratoriosSilanes S.A. of C.V. and entitled: “Imunógeno e antiveneno contra oveneno da aranha marrom” describes the isolation, characterization andexpression of coding DNA fragments of sphingomyelinases D of 3 speciesof spiders of the genus Loxosceles: L. boneti, L. reclusa and L. laetaand its use as an immunogen for the production of neutralizingantibodies of the corresponding venom and of respective F(ab')2fragments. Said document PI 0514809-0 also reports the use ofrecombinant sphingomyelinases D as part of an antigenic array useful inimmunopurifying antibodies and its fragments as part of a diagnosticdevice for venoming by spider of the genus Loxosceles.

As can be observed, document PI 0514809-0 basically describes theproduction of recombinant sphingomyelinases for producing neutralizingantibodies against spider venom of the genus Loxosceles and its use incompositions for treating venoming by these spiders. In no time documentPI 0514809-0 describes or suggests the use of inhibitor compoundsbelonging to the class of benzenesulfonamides andchloro-benzenesulfonates for treating symptoms associated withloxoscelism caused by the bite of spiders belonging to genus Loxosceles.

The international patent application WO 01/74343, filed on Mar. 30,2001, published on Oct. 11, 2001; on behalf of 3M INNOVATIVE PROPERTIESCOMPANY and entitled: “Method for the treatment of dermal lesions causedby envenomation” relates to a method of treating skin lesions caused byvenoming, comprising the application, at the site of injury, of atherapeutically effective amount of a modifier compound of the immuneresponse selected from the group consisting of imidazoquinoline amines,imidazopyridine amines, 6.7-fused cycloalkyl imidazopyridine amines,imidazonaphtiridine amines, tetra-hydro imidazonaphtiridine amines,oxazolopyridine amines, oxazoloquinoline amines, thiazolopyrimidineamines, thiazoloquinoline amines and 1.2-bridged imidazoquinolineamines.

As noted, the international patent application WO 01/74343 only providesthe use of known immune response modifier (IRMs) compounds mentionedabove, which are able to stimulate the innate and acquired immuneresponse for the treatment of skin lesions by venoming caused, forexample, by bites of spiders of the genus Loxosceles, among others. Saidinternational patent application WO 01/74343 neither mentions norsuggests the use of specific inhibitor compounds to sphingomyelinases(D) present in the venom of spiders of the genus Loxosceles for thetreatment of skin lesions and/or to avoid developing loxoscelism.

The international patent application WO 2007/149343, filed on Jun. 15,2007, published on Dec. 27, 2007; on behalf of THE BOARD OF TRUSTEES OFTHE LELAND STANFORD JUNIOR UNIVERSITY and entitled: “Proteases fortreatment of venemous bites” relates to materials and methods, includingkits, for use in the treatment of snake bites, bee stings, spider bitesand other forms of venoming or exposure to toxins. The materials andmethods involve the use of proteases associated with the protectivemechanism of mast cell degranulation, associated with a reduction in thetoxic effects and increased survival rate in animal models. Theproteases used are selected from the group containing chymases(specificity to a substrate similar to chymotrypsin), carboxypeptidaseA, carboxypeptidase B, tryptases (specificity to a substrate similar totrypsin), chymotrypsin or papain.

Although the international patent application WO 2007/149343 uses, inits embodiment examples, treatment for venoming caused by spiderLoxosceles reclusa, said international patent application WO 2007/149343does not provide for the use of a specific inhibitor compound forsphingomyelinase D of spiders of the genus Loxosceles.

The international patent application WO 2008/022771, filed on Aug. 21,2007, and published on Feb. 28, 2008; on behalf of NOVARTIS A G andentitled: “Amides as sphingomieline inhibitors” describes a formulationthat provides the use of acid sphingomyelinase for treating disordersmediated by the activity of acid sphingomyelinase present in thelysosome of mammalian cells, such as autoimmune diseases, diseasesinvolving abnormal apoptosis and tumor growth. Therefore, said documentdoes not describe any compounds that can specifically inhibit thetoxicity of sphingomyelinases D from the venoms of spiders of the genusLoxosceles, controlling the development of cutaneous and systemicloxoscelism.

The U.S. patent application US 2010/0099881 A1 (which corresponds to theinternational application PCT/JP2008/053936), filed on Dec. 7, 2009, andpublished on Apr. 22, 2010; on behalf of Mugio Nishizawa, HiroshiImagawa, Jun Sakurai, Masataka Oda and Otsuka Chemical Co. LTD. andentitled: “Sphingosine compound, method for producing the same, andsphingomyelinase inhibitor”, only discloses a new sphingosine compound,with inhibitory activity of sphingomyelinase suitable for use as amedicine for treating or preventing cerebral hemorrhage, cerebralinfarction and similar cerebrovascular diseases, head injuries, seniledementia, Alzheimer's and Parkinson's disease, among other similardegenerative diseases, diabetes, obesity, atherosclerosis, inflammatorydiseases, autoimmune diseases, cancer, kidney disease and heartdiseases.

The article published in Journal of Investigative Dermatology (2007),volume 127, pages 1410 to 1418, available online on Jan. 11, 2007,Authors: Danielle Paixão-Cavalcante, Carmem W. van der Berg, Rute M.Gonçalves-de-Andrade, Matheus de F. Fernandes-Pedrosa, Cinthya KimoriOkamoto e Denise V. Tambourgi, entitled: “Tetracicline protects againstdermonecrosis induced by loxosceles spider venom”, relates to the use ofclassic tetracycline antibiotics (tetracycline, doxycycline andminocycline) in the inhibition or reduction of dermonecrotic lesions andto the mechanisms involved in the development of cutaneous loxoscelisminduced by the venom of Loxosceles spiders through in vitro and in vivoexperiments. According to this document, the topical treatment is moreeffective in the prevention or reduction of dermonecrotic lesions thanthe oral (systemic) treatment, possibly by the concentration oftetracyclines used which, in high systemic concentrations, can lead totoxicity. According to said document, the binding of sphingomyelinase Dfrom the venom of Loxosceles spiders to the cell surface induces theexpression and activation of endogenous metalloproteinases (MMPs). Thus,the mechanism of action proposed in the document involves thetetracycline inhibitory action on metalloproteinases (MMPs), whoseexpression and activation are induced by the venom of Loxoscelesspiders. In no time said document refers to the use of compoundsbelonging to the class of benzenesulfonamides andchloro-benzenesulfonates with similar purposes.

As can be observed, there is no state of the art document that describesor suggests the use of inhibitor compounds belonging to the class ofbenzenesulfonamides and benzenesulfonates for treating symptomsassociated with loxoscelism caused by the bite of spiders belonging togenus Loxosceles.

Dictionary—See detailed dictionary

SUMMARY OF THE INVENTION

In order to solve the problems mentioned above, this invention providessignificant advantages related to the use of inhibitor compoundsbelonging to the class of benzenesulfonamides and benzenesulphonate fortreating symptoms associated with loxoscelism caused by the bite ofspiders belonging to genus Loxosceles, enabling an increase in theirperformance and offering a more favorable cost/benefit ratio.

This invention relates to the use of inhibitors compounds belonging tothe class of benzenesulfonamides and benzenesulphonate with inhibitoryactivity on sphingomyelinases D of the venom of Loxosceles spiders forpreparing a medicine to act on the hydrolytic activity of recombinantsphingomyelinase D toxin (SMase D) and of the venom of brown reclusespider Loxosceles laeta.

The present invention preferably relates to the use of4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamideand of4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate(compounds 5 and 6, respectively), which are compounds that can inhibitthe toxicity action of sphingomyelinases D of the venom of Loxosceles,controlling the development of cutaneous and systemic loxoscelism;reduction of hemolysis, inhibition of cutaneous lesion, inhibition ofdermonecrosis, inhibition of the intracellular signaling pathways andproduction of reactive oxygen species. Besides of the therapeuticpotential, such inhibitors can be used as tools in the study of theaction of sphingomyelinases and phospholipases D.

In a second aspect, this invention relates to a pharmaceuticalcomposition for treating loxoscelism, hemolysis reduction, inhibition ofcutaneous lesion, inhibition of dermonecrosis, inhibition ofintracellular signaling pathways and production of reactive oxygenspecies, which comprises a benzenesulfonamide compound and apharmaceutically acceptable vehicle. The benzenesulfonamide compoundpreferably being the compound4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamine]benzenesulfonamide.

A third aspect of this invention relates to a pharmaceutical compositionfor treating loxoscelism, hemolysis reduction, inhibition of cutaneouslesion, inhibition of dermonecrosis, inhibition of intracellularsignaling pathways and production of reactive oxygen species, whichcomprises a benzenesulphonate compound and a pharmaceutically acceptablevehicle. The benzenesulphonate compound preferably being compound4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and operation of the invention, along with the additionaladvantages thereof, can be better understood by reference to theattached drawings and the description that follows:

FIG. 1 shows the structural formula of compound5—4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide;

FIG. 2 shows the structural formula of compound6—4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate;

FIG. 3 shows a graphic indicating the inhibition ability of thehydrolytic activity of recombinant sphingomyelinases and present in thevenom of Loxosceles laeta, on the substrate sphingomyelin (SM), bycompounds 5 and 6;

FIG. 4 shows a graphic indicating the inhibition ability of thehydrolytic activity of recombinant sphingomyelinases and present in thevenom of Loxosceles laeta, on the substrate lysophosphatidylcholine(LPC), by compounds 5 and 6;

FIGS. 5 (a) and 5 (b) show the dose-response curves of compounds 5 and 6in the inhibition of the hydrolytic activity on SM, on which basis theIC₅₀ values of compounds have been calculated;

FIG. 6 presents the inhibition percentage of removal of glycophorin Cfrom the surface of red blood cells by compounds 5 and 6, a crucialevent in the development of complement system-dependent hemolysisobserved in venoming;

FIG. 7 shows the inhibition percentage of the sphingomyelinases Dbinding to the surface of red blood cells, by compounds 5 and 6;

FIGS. 8 and 9 represent the ability of compounds 5 and 6 to reduce thedeath of human keratinocytes induced by recombinant sphingomyelinasespresent in the venom of L. laeta;

FIG. 10 shows the inhibition percentage of the binding of recombinantsphingomyelinases present in the venom to the cell membrane of humankeratinocytes, by compounds 5 and 6;

FIG. 11 describes the inhibition percentage of production/secretion ofmatrix metalloproteinases (MMP-2 and 9) by human keratinocytes treatedwith recombinant sphingomyelinases D promoted by compounds 5 and 6;

FIG. 12 shows the inhibition percentage of the development ofdermonecrotic lesions in rabbits, analyzed 24, 48 and 72 hours afterinoculation of the venom, by compounds 5 and 6;

FIG. 13 represents the inhibition percentage of MAPK ERK1/2intracellular signaling pathway activation in human keratinocytestreated with recombinant sphingomyelinases D by compounds 5 and 6;

FIG. 14 shows the inhibition percentage of superoxide ion production byhuman keratinocytes treated with sphingomyelinases D recombinant, bycompounds 5 and 6;

FIG. 15 shows the inhibition percentage of the production of cytokinesTNF-α and TGF-β1 by human keratinocytes treated with the recombinantsphingomyelinases D, by compounds 5 and 6;

FIG. 16 shows the average inhibition of all activities tested forcompounds 5 and 6;

FIG. 17 shows the action of inhibitor compounds 5 and 6 on the substratehydrolytic activity of PLD of Corynebacterium pseudotuberculosis;

FIG. 18 shows the action of inhibitor compounds 5 and 6 on thehydrolytic activity on sphingomyelin substrate, of PLD of Bacilluscereus; and

FIG. 19 shows the action of inhibitor compounds 5 and 6 on thehydrolytic activity on sphingomyelin substrate, of PLD of Staphylococcusaureus.

Annex 1 shows microphotographs of histopathological analysis of the skinof rabbits inoculated with the venom of L. laeta or with the venomincubated with compounds 5 and 6.

DETAILED DESCRIPTION OF THE INVENTION

Although this invention can be susceptible to different embodiments, apreferred embodiment is shown in the drawings and in the followingdetailed discussion, with the understanding that this embodiment shouldbe considered an example of the principles of the invention without theintention of limiting the present invention to what has been illustratedand described herein.

This invention relates to the use of inhibitors compounds belonging tothe class of benzenesulfonamides and benzenesulphonate with inhibitoryactivity on sphingomyelinases D of the venom of Loxosceles spiders forpreparing a medicine to act on the hydrolytic activity of recombinantsphingomyelinase D toxin (SMase D) and of the venom of brown reclusespider Loxosceles laeta.

The present invention preferably relates to the use of4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide,named compound 5 (FIG. 1) and of4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate,and compound 6 (FIG. 2), which are compounds that can inhibit thetoxicity of sphingomyelinases D of the venom of Loxosceles, controllingthe development of cutaneous and systemic loxoscelism, treatment ofloxoscelism, reduction of hemolysis, inhibition of cutaneous lesion,inhibition of dermonecrosis, inhibition of the intracellular signalingpathways and production of reactive oxygen species. Besides of thetherapeutic potential, such inhibitors can be used as tools in the studyof the action of sphingomyelinases and phospholipases D.

The inhibitor compounds of the present invention can be used as tools inthe study of the action of sphingomyelinases and phospholipases D, andthese inhibitors have therapeutic potential to venoming by Loxoscelesspiders and infections by SMases producer microorganisms.

Selection of Compounds

The binders used in molecular Docking studies were selected from a freedatabase of commercially available molecules, the ZINC Database(www.zinc.docking.org). Many ligands in this database are available in avariety of 3D formats immediately usable by many popular dockingprograms.

In addition to the important physical-chemical properties, the bankcontains supplier information and numbers of the original catalogue foreach commercial source of this compound. It is also possible to findinformation about the function or activities already studied for thecompound, when available.

Molecular Docking Studies

Molecular docking studies for selecting SMases D inhibitors wereconducted at the Laboratório Nacional de Luz Síncroton, Campinas—SaoPaulo, with the aid of ICM software—Molsoft (based on Monte Carloalgorithm) Molegro Virtual Docker, (based on a heuristic searchalgorithm that combines differential evolution with a cavity predictionalgorithm) and GOLD (based on a genetic algorithm). The docking analysiswere carried out based on the 3D structure of SMase I of the venom of L.laeta, according to article of MURAKAMI, M. T.; FERNANDES-PEDROSA, M.F.; TAMBOURGI, D. V.; ARNI, R. K. and entitled “Structural basis formetal ion coordination and the catalytic mechanism of sphingomyelinasesD”, published in the J. Biol. Chem., v. 280, No. 14, page 13658-13664,2005, and incorporated herein by reference.

The following topics describe the action of compounds4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide(FIGS. 1) and4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulphonate(FIG. 2), named as compounds 5 and 6 of this invention, in themechanisms that underlie the development of cutaneous and systemicloxoscelism.

Analysis of the Inhibitory Action of Compounds 5 and 6 on the HydrolyticActivity of the Toxin and Venom.

Compounds 5 and 6 of this invention were diluted in dimethyl sulfoxide(DMSO) and prepared in a stock solution of high concentration, which wasdiluted in sterile saline solution to the concentrations used in eachassay.

In the fluorimetric assays, the basal fluorescence of the compounds wasmeasured and deducted from these assays.

The ability of the compounds in the concentration of 40 μM of inhibitingthe hydrolytic activity of the recombinant toxin sphingomyelinase D(SMase D) and of the venom of Loxosceles laeta on substratessphingomyelin (SM) and lysophosphatidylcholine (LPC), by fluorimetryassays, was analyzed.

FIG. 3 shows the inhibition percentage of compounds 5 and 6 on thehydrolytic activity of recombinant SMases D and present in the venom ofL. laeta, on the sphingomyelin (SM) substrate.

The results showed that compound 5 of the present invention inhibits45.7% and 53% of the activity of recombinant toxin and the venom of L.laeta, respectively, on the SM substrate. Compound 6 of the presentinvention inhibited 51% and 22.7% of the activity of the recombinanttoxicity and the venom, respectively.

FIG. 4 shows the inhibition percentage of compounds 5 and 6 on thehydrolytic activity of recombinant SMases D and present in the venom ofL. laeta, on the lysophosphatidylcholine substrate (LPC).

In relation to substrate LPC, compound 5 of the present inventionreduces 38.6% and 44.4% of the activity of the recombinant toxin and thevenom, respectively. As to compound 6 of this invention, it reduces 34and 16% of this activity.

FIGS. 5(a) and 5(b) show the dose-response curves of the inhibition ofthe hydrolytic activity of recombinant SMases D on the substratessphingomyelin, respectively by compounds 5 and 6. Based on these curves,IC₅₀ of the compounds were calculated.

Based on said dose-response curves of the action of the compounds on theactivity of the recombinant toxin on the substrate SM, the IC₅₀ valueswere obtained, which were 45.4±1.2 μM to compound 5 of this inventionand 63.4±1.1 μM to compound 6 of this invention.

The analysis of the inhibition mechanism showed that compounds 5 and 6of the present invention may be classified as uncompetitive actioninhibitors with K_(i) values of 1.63 and 1.73, respectively.

Analysis of the Action of Compounds 5 and 6 of the Present Invention onHemolysis Induced by the Venom of Loxosceles

The removal of glycophorins from the surface of red blood cells byindirect action of the toxin is a crucial event for the development ofintravascular hemolysis that occurs in accidents.

FIG. 6 shows the inhibition percentage of the removal of glycophorins Cof the surface of human red blood cells induced by SMases D present inthe venom of L. laeta.

The analysis of the expression of glycophorin C on the surface of humanred blood cells by flow cytometry showed that, in the presence ofcompounds 5 and 6 of the present invention (40 μM), the removal of thesemolecules from the surface of cells is reduced by 92.6 and 88.2%,respectively.

FIG. 7 shows a graph indicating the inhibition percentage of the bindingof SMases D present in the venom of L. laeta to the surface of human redblood cells.

This event of glycophorin C removal is associated to the binding of thetoxin on the red blood cell membrane. Thus, the binding of the toxin tothe cell surface was analyzed by flow cytometry and the resultsindicated a reduction of 96.8 and 84% in the presence of the compounds 5and 6 of the present invention, respectively.

Analysis of the Action of Compounds 5 and 6 of the Present Invention onthe Mechanisms of Cutaneous Loxoscelism

The development of cutaneous lesion observed in the loxoscelism isclosely related to the cell death of keratinocytes induced by SMase D.

FIG. 8 shows the viability of human keratinocytes treated only withrecombinant SMase D or incubated with the compounds 5 and 6. Theanalysis of cell viability by MTT method in vitro, after treatment withrecombinant toxin or venom, showed that in the presence of the compound5 of the present invention the viability of cells treated with the toxinincreases in 20.23 to 55.75% and in the presence of the compound 6 ofthe present invention increases to 53.55%.

FIG. 9 shows the viability of human keratinocytes treated only withvenom of L. laeta or with the venom incubated with compounds 5 and 6.The cells treated with venom had increased viability of 36.7 to 45.9%,with 5 of the present invention, and 59.26% with compound 6 of thepresent invention. For these assays, compounds 5 and 6 of this inventionwere used at a concentration of 10 μM.

As well as in erythrocytes, keratinocyte cell death is associated withthe binding of the toxin to this cell membrane.

FIG. 10 shows a graph indicating the inhibition percentage of thebonding of recombinant SMases D and present in the venom of L. laeta tothe surface of human keratinocytes. The analysis of this parameter byflow cytometry showed that the binding of recombinant toxin is reducedby 54.9% with compound 5 of this invention and 50.77% with compound 6 ofthis invention, both at a concentration of 40 μM. The binding of SMasespresent in venom is reduced by 63.8 and 25.6% in the presence ofcompounds 5 and 6, respectively.

Other event associated with keratinocyte death during the development ofskin lesion is the production of extracellular matrix metalloproteinases2 and 9 (MMPs). Thus, the culture supernatant of keratinocytes treatedwith the venom of L. laeta was investigated by ELISA as for the presenceof MMP-2 and 9. In the presence of compounds 5 and 6 of the presentinvention (10 μM), the MMP-2 secretion is reduced by 81 and 98.4%,respectively. In relation to MMP-9, both compounds completely inhibitthe secretion of this MMP.

FIG. 11 shows a graph indicating the inhibition percentage ofproduction/secretion of MMP-2 and 9 by human keratinocytes treated withthe venom of L. laeta.

After analyzing the aspects involving the development of skin lesion,the ability of the compounds in inhibiting dermonecrosis in vivo wasverified, using a model in rabbits.

After 24 hours of venom inoculation, the injury was reduced by 61.8 and36% in the presence of compounds 5 and 6 of the present invention,respectively. In 48 hours, the inhibition was 60 and 45%, and in 72hours, it was 56 and 49% in the presence of these two compounds.

FIG. 12 shows a graph indicating the inhibition percentage ofdermonecrotic lesions developed in rabbits by inoculation of the venomof L. laeta.

The histopathological analysis of the skin of rabbits inoculated withthe venom in the presence or absence of these compounds demonstratesthat, in the presence of compounds 5 and 6 of the present invention,there is a reduction in the disorganization of the collagen fibers inthe dermis, absence of hemorrhage and inflammatory infiltrate, as wellas injury to the adjacent muscle layer in relation to that inoculatedonly with venom.

Annex 1 shows microphotographs of the histopathological analysis of theskin of rabbits inoculated only with the venom of L. laeta or with thevenom incubated with compounds 5 and 6.

Compounds 5 and 6 of this invention were used at a concentrationcorresponding to three times the value of IC₅₀ (136.2 μM to compound 5and 190.2 μM to compound 6).

Action of Compounds 5 and 6 of the Present Invention on Other MechanismsInvolved in the Toxicity of Sphingomyelinases D of the Venom ofLoxosceles

FIG. 13 shows the percentage of inhibition of the activation of MAPKERK1/2 intracellular signaling pathway induced by recombinant SMases D.The toxin was able to induce the intracellular MAPKs intracellularsignaling pathway, more specifically, the ERK1/2 in keratinocytes. Itwas verified, in ELISA assays, that in the presence of compounds 5 and 6of this invention (40 μM) the phosphorylated ERK1/2 was reduced by 65.8and 80.2%, respectively.

Another aspect analyzed was the production of reactive oxygen species bykeratinocytes treated with the toxin, using the flow cytometrytechnique.

FIG. 14 shows the inhibition percentage of the production of superoxideion by keratinocytes treated with recombinant SMases D.

The results indicate that, in the presence of compounds 5 and 6 of thepresent invention (40 μM), superoxide production by these cells wasinhibited by 70.7 and 92.7%, respectively.

Additionally, the expression of the TNF receptor on the surface ofkeratinocytes treated with the toxin was analyzed by flow cytometry. Thetreatment with the toxin leads to a removal of the cell surfacereceptor, which is reversed by 27.7% in the presence of compound 5 ofthis invention (40 μM).

The production of cytokines by keratinocytes can be an important aspectin the development of skin lesion of loxoscelism.

FIG. 15 shows the inhibition percentage of the production of cytokinesby human keratinocytes treated with the recombinant SMases D. It wasdemonstrated, by ELISA, on the culture supernatant of keratinocytestreated with the toxin, that the same induces the production of TNF-α,which was reduced by 73.9 and 61.1% in the presence of compounds 5 and 6(10 μM), respectively. Another cytokine found was

TGF-β1, which was reduced by 88.3 and 89.2% in the presence of thecompounds.

FIG. 16 shows a graphic indicating the efficiency of compounds 5 and 6in the inhibition of the mechanisms involved in the toxicity of SMases Dand in the development of cutaneous and systemic loxoscelism.

Thus, taking into consideration all the aspects analyzed, compound 5 ofthe present invention is 61.1% efficient, while compound 6 of thisinvention is 54.1% efficient in relation to the mechanisms involved inthe toxicity of SMases D in the development of loxoscelism.

Action of Inhibitor Compounds 5 and 6 on the Hydrolytic Activity onSubstrate Sphingomyelin, of Bacterial Sphingomyelinases.

Phospholipases are important toxic components present in the venom ofanimals and bacterial toxins. They promote the hydrolysis of ester bondsof phospholipids, and they are classified in phospholipases A1, A2, Cand D, by the position of hydrolyzed ester bond (VAN DEN BOSCH, 1980).

Contrary to other phospholipases, those found in the venom of Loxosceles(SMases D) and in certain bacteria have an unusual substratespecificity, since of the four major phospholipids present in mammalcell membranes, only sphingomyelin is hydrolyzed by bacterial LDP, whilethose of the venom of Loxosceles spiders cleave sphingomyelin,generating ceramide-1-phosphate as the hydrolysis product and are alsoable to hydrolyze lysophosphatidylcholine, generating lysophosphatidicacid (Bernheimer; Campbell; Forrester, 1985; Tambourgi et al., 1998; VanMeeteren et al., 2004). Researches on sequence databases showed that thetoxins of the bacteria Corynebacterium pseudotuberculosis andArcanobacterium haemolyticum are also sphingomyelinases and have between24 and 34% of similarity with the first 30 amino acids of the Loxoscelestoxins (Tambourgi et al., 1998), as well as similar molecular weight andisoelectric point (Bernheimer; Campbell; Forrester, 1985); moreover, thetoxins of these bacteria induce biological effects also similar to thoseinduced by the venom of Loxosceles (Bernheimer; Campbell; Forrester,1985; Forrester; Barrett; Campbell, 1978; McNamara; Cuevas; Songer,1995; Rees et al., 1984; Tambourgi et al., 1998, 2002, 2007).

Corynebacterium pseudotuberculosis, Corynebacterium ulcerans andArcanobacterium haemolyticum are pathogens of pets and humans (McNAMARAet al., 1995).

C. pseudotuberculosis is a gram positive Bacillus widely distributed inanimal populations, causing caseous lymphadenitis in sheep, goats andboth, ulcerative inflammation of the wall of the lymphatic vessels andpectoral, abdominal and inguinal abscesses in horses (Soger et al.,1990; Huerta et al., 2013). Infections also occur in cattle and humans.These infections lead to reduced production and milk quality in cattleand goats and to a low yield of wool in sheep (Hoelzle et al., 2013). Inthe mechanism of pathogenesis of bacteria C. pseudotuberculosis,sphingomyelinase (PLD) has been shown as an essential determinant ofvirulence, which contributes to bacteria spreading from the initial siteof infection to secondary host sites (Nairn et al., 1977). Knockout PLDstrains have shown reduced virulence, emphasizing the importance of thistoxin in the pathogenesis (Hodgson et al., 1992; McNamara et al., 1994).

Arcanobacterium haemolyticum are gram-positive bacteria responsible formany respiratory infections in healthy people. Most cases involvepharyngitis and/or tonsillitis, and approximately 50% are exsudative.Throat infections are often accompanied by cervical lymphadenopathy(GREEN et al., 1981; MACKENZIE et al., 1995). This bacterium produces aPLD which acts preferentially on sphingomyelin, generatingceramide-1-phosphate in the target membrane and being closely related tothe PLD produced by C. pseudotuberculosis (Linder, 1997).

Corynebacterium ulcerans has its main reservoir in cattle herd, in whichit induces mastitis, however, cases in human patients have been reported(DIAS et al., 2009). This bacterium can produce diphtheria toxin (DT)and/or PLD, and can cause infections in humans, mimicking the cutaneousand the classic respiratory diphtheria with pseudomembranas (Dewinter etal., 2005; Tiwari et al., 2008). Furthermore, the C. ulcerans strainsthat produce PLD, but not DT, also affect the lower respiratory tractand are capable of causing severe disease in humans, such as pneumonia(Mane-Guaraldi et al., 2008) and granulomatous nodules in lungs (Deseauet al., 1995).

Similarities in physical properties and activities of PLDs produced byC. pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticumsuggest that this group of enzymes is important in the pathogenesis ofdiseases caused by these organisms. Thus, considering such similaritiesin PLD activities and clinical conditions induced by these bacteria andby Loxosceles toxins, it is important to test the compounds withactivity on the Loxosceles SMases D on the PLD activity of some of thesebacteria in order to possibly obtain compounds that help in thetreatment of these diseases that affect farm animals and humans.

for this, 50 μg of PLD of C. pseudotuberculosis incubated with 40 μM ofinhibitor compounds 5 and 6, and the residual hydrolytic activity onsphingomyelin were evaluated by fluorimetry. The results showed that thePLD activity of C. pseudotuberculosis was reduced about 70% by compound5 and about 68% by compound 6 (FIG. 17).

Another class of phospholipases produced by bacteria and related to thevirulence of the same are phospholipases C (PLC). Bacteria such asStaphylococcus aureus, Bacillus cereus, Leptospira and Listeria, amongothers, are some of the PLC producers.

Bacillus cereus is well known for its role as a mediator of foodbornediseases (Taylor & Gilbert, 1975; Gianella & Brasile, 1979; Stenfors etal., 2008; Bottone, 2010). This organism form spores and is widelydistributed in dust, air and water, and therefore, it is ubiquitous inhospital environments, infecting clothings and intravenous catheters,thus providing an opportunity for infection in immunocompromisedpatients (Drobniewski, 1993). Generally, the infection caused by thisorganism leads to mild gastroenteritis, however, outbreaks of deadlyfood poisoning by B. cereus (Lund et al., 2000; DIERICK et al., 2005)and local and systemic infections out of the gastrointestinal tract(endophthalmitis, pneumonia and sepsis) in humans have been reported(Bottone, 2010).

B. cereus secrets several toxins that target cell membranes, includingBc-SMases. Evidences point out that, in Listeria ivanovii, PLC can acton the exit of the bacteria inside the phagocytic vacuoles, thuspromoting the intracellular survival and spread of the pathogen(Gonzalez-Zorn et al., 1999). SMase derived from Helicobacter pyloridirectly contributes to the cytotoxicity on gastric cells (Tseng et al.,2004) and toxins with SMase activity are expressed by 91% of theStaphylococcus aureus strains of high toxicity (Collins et al., 2008).

Recent studies have shown that Bc-SMases lyse sheep red blood cellscontaining large amounts of sphingomyelin in the membrane (Oda et al.,2010), that these belong to the same group of SMases from H. pylori andL. ivanovii (Openshaw et al., 2005) and that the hydrolysis ofsphingomyelin in the membrane of macrophages generates ceramide, whichmitigates the fluidity of the membrane, leading to an unsuccessfulphagocytosis. Therefore, PLC or Bc-SMase plays a key role in the bypassof the bacteria on immune mechanisms in macrophages (Oda et al., 2012).

S. aureus produces and secretes a large number of cell surface virulencefactors that enable this organism to cause a variety of human diseasesranging from relatively mild eruptions and subcutaneous abscesses tosevere septic shock and necrotizing pneumonia (Lowy, 1998; McCormick etal., 2001), moreover, S. aureus was also found in cases of bovinemastitis (Aarestrup et al., 1999). One of the exotoxins produced is abeta-toxin with mass about 35 kDa which seems to work like a SMase,being classified as a PLC (Huseby et al., 2007). This toxin causes lysisof red blood cells and other cells, such as lymphocytes and neutrophils(Marshall et al., 2000), being found in 72% of the cases of bovinemastitis, 11% in bacteria isolated from nostrils of healthy people andin 13% of cases of septic shock (Aarestrup et al., 1999). In general,studies suggest that this toxin can also contribute to immunomodulationof the host in the presence of other virulence factors (HUSEBY et al.,2007).

Thus, knowing the importance of PLCs in infections by some bacteria,compounds 5 and 6 (40 μM) were tested in face of the hydrolytic activityof SMases of B. cereus and Stahylococcus aureus. The results showed thatthe activities of SMases from both bacteria were efficiently reduced byincubation with compounds 5 and 6 (FIGS. 18 and 19).

Besides of the cited bacteria, other bacteria highly pathogenic toanimals and to men, such as Leptospira interrogans (Kasarov & Addamiano,1969; Nayaranavari et al., 2012), Listeria ivanovii (Gonzalez-Zorn etal., 1999), Helicobacter pylori (Tseng et al., 2004) and Pseudomonas sp(Sueyoshi et al., 2002), among others, also produce PLDs or PLCs, whichusually are part of the virulence mechanisms. In addition to bacteria,parasites such as Clonorchis sinensis (Huang et al., 2013), ticks of thegenus Ixodes (Alarcon-Chaidez et al., 2009), and fungi of the genusAspergillus and Coccidioides (Fry et al., 2009) also produce suchmolecules.

Therefore, based on the results presented herein and on the similarityof the physical properties and biological activities between SMases ofLoxosceles and of bacteria and other organisms, we propose here that thecompounds 5 and 6, selected through molecular docking studies on thetertiary structure of SMase D I of Loxosceles laeta, can be used astools to study the virulence mechanisms of these organisms, as well as apossible basis for the development of complementary therapeuticstrategies for the control of diseases caused by these organisms.

Therefore, compounds 5 and 6 of the present invention can be used:

-   -   For preparing a medicine to act on the hydrolytic activity of        recombinant toxin sphingomyelinase D (SMase D) and of the venom        of brown recluse spider Loxosceles laeta;    -   For preparing a medicine to act on hemolysis reduction,    -   For preparing a medicine to inhibit skin lesion,    -   For preparing a medicine to inhibit dermonecrosis,    -   For preparing a medicine to inhibit mechanisms involved in the        toxicity of SMases D in the development of loxoscelism;    -   For preparing a medicine to inhibit intracellular signaling        pathways and the production of reactive oxygen species

Besides of the fact that compounds 5 and 6 of the present invention actas inhibitors of sphingomyelinases D of the venom of spiders of thegenus Loxosceles, they can also act as a complimentary therapeutic drugfor accidents and possibly as compounds for controlling the effects ofbacterial sphingomyelinases (Coryneumbacterium pseudotuberculosis,Arcanobacterium haemoliticum, Bacillus cereus) and some arthropods(spiders, scorpions and ticks).

In a second aspect, this invention relates to a pharmaceuticalcomposition for treating loxoscelism, hemolysis reduction, inhibition ofcutaneous lesion, inhibition of dermonecrosis, inhibition ofintracellular signaling pathways and production of reactive oxygenspecies, which comprises a benzenesulfonamide compound and apharmaceutically acceptable vehicle. The benzenesulfonamide compoundpreferably being the compound4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamine]benzenesulfonamide.

A third aspect of this invention relates to a pharmaceutical compositionfor treating loxoscelism, hemolysis reduction, inhibition of cutaneouslesion, inhibition of dermonecrosis, inhibition of intracellularsignaling pathways and production of reactive oxygen species, whichcomprises a benzenesulphonate compound and a pharmaceutically acceptablevehicle. The benzenesulphonate compound preferably being compound4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.

It is understood, by “pharmaceutically acceptable vehicle” anyacceptable vehicle, excipient or diluent in the pharmaceutical field.

Thus, although only some details of the present invention have beenshown, it will be understood that several omissions, substitutions andchanges to classes of benzenesulfonamide and benzenesuphonate compoundscan be made by a person skilled in the art, without departing from thespirit and scope of this invention.

It is expressly provided that all combinations of the elements thatperform the same function substantially the same way to achieve the sameresults are within the scope of the invention. Substitution of elementsin an embodiment described to another are also fully comprised andcontemplated.

It should be also understood that the drawings are not necessarily inscale, and are only conceptual in nature. The intention is, therefore,to be limited, as indicated by the scope of the attached claims.

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1. A pharmaceutical composition comprising (1) a compound selected fromthe group consisting of a benzenesulfonamide compound and abenzenesulphonate compound and (2) a pharmaceutically acceptablevehicle.
 2. (canceled)
 3. The pharmaceutical composition, of claim 1,wherein the compound is the benzenesulfonamide compound, wherein thebenzenesulfonamide compound is4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methylene amine]benzenesulfonamide.
 4. The pharmaceutical composition, of claim 1,wherein the compound is the benzenesulphonate compound, wherein thebenzenesulphonate compound is preferably compound4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.5. The pharmaceutical composition of claim 1, which inhibits thetoxicity of D sphingomyelinases in the venom of Loxosceles spiders. 6.(canceled)
 7. The pharmaceutical composition of claim 5, wherein thecompound is the benzenesulfonamide compound and wherein thebenzenesulfonamide compound is4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenosulfonamide.8. The pharmaceutical composition of claim 5, wherein the compound isthe benzenesulphonate compound, wherein the benzenesulphonate compoundis4-methyl-3-oxo-2-(3-pyridylmethylene)benzo[3,4-b]furan-6-yl-4-chlorobenzenesulfonate.9. The pharmaceutical composition of claim 5, wherein the compound actson the hydrolytic activity of recombinant toxin sphingomyelinase D (DSMase) and of venom of brown recluse spider.
 10. The pharmaceuticalcomposition of claim 5, wherein the compound controls the development ofcutaneous and systemic loxoscelism.
 11. (canceled)
 12. (canceled) 13.The pharmaceutical composition of claim 7, wherein the4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenesulfonamide: (1) inhibits by 45.7% the activity ofrecombinant toxin D SMase on a sphingomyelinase substrate, (2) inhibitsby 53% the activity of the venom of L. laeta on SM substrate, (3)reduces by 38.6% the activity of recombinant toxin D SMase in relationto lysophosphatidylcholine (LPC) substrate, (4) reduces by 44.4% thevenom activity in relation to LPC substrate, (5) has an IC₅₀ value of45.4±1.2 μM, based on action of the compound on the activity ofrecombinant toxin on SM substrate, (6) reduces by 92.6% the removal ofglycophorins from the surface of red blood cells, (7) reduces by 96.8%the removal of glycophorin C from the surface of red blood cells, (8)increases from 20.23% to 55.75% the viability of cells treated with therecombinant toxin D SMase, (9) increases from 36.7% to 45.9% theviability of cells treated with the venom, (10) reduces by 81% secretionof extracellular matrix metalloproteinases 2 (MMP-2) in keratinocytestreated with recombinant toxin D SMase, (11) completely inhibitssecretion of extracellular matrix metalloproteinases 9 (MMP-9) inkeratinocytes treated with recombinant toxin D SMase, (12) reducesdisorganization of dermis collagen fibers, bleeding, inflammatoryinfiltrate, and injury to adjacent muscle layer in skin contacted withvenom, (13) inhibits by 70.7% superoxide production by keratinocytestreated with recombinant toxin D SMase, (14) reverses by 27.7% theremoval of TNF receptor from the surface of keratinocytes treated withrecombinant toxin D SMase, (15) reverses by 73.9% the production ofTNF-α in keratinocytes treated with recombinant toxin D SMase, (16)reduces by 88.3% the production of TGF-β1 in keratinocytes treated withrecombinant toxin D SMase, or (17) reduces by 65.8% phosphorylatedERK1/2 in keratinocytes treated with recombinant toxin D SMase.
 14. Thepharmaceutical composition of claim 8, wherein the4-bromo-N-[(E)-(2-methyl-1H-indol-3-yl)methyleneamino]benzenesulfonamide; (1) inhibits by 51% the activity ofrecombinant toxin D SMase on SM substrate, (2) inhibits by 22.7% theactivity of the venom of L. laeta on SM substrate, (3) reduces by 34%the activity of recombinant toxin D SMase in relation to LPC substrate,(4) reduces by 16% the activity of the venom in relation to substrateLPC, (5) has an IC₅₀ value is 63.4±1.1 μM, based on the action of thecompound on the activity of recombinant toxin on SM substrate, (6)reduces by 88.2% the removal of glycophorins from the surface of redblood cells, (7) reduces by 84% the removal of glycophorin C from thesurface of red blood cells, (8) increases from 20.23% to 53.55% theviability of cells treated with recombinant toxin D SMase, (9) increasesfrom 36.7 to 59.26% the viability of cells treated with the venom, (10)reduces by 98.4% the secretion of extracellular MMP-2 in keratinocytestreated with recombinant toxin D SMase, (11) completely inhibits thesecretion of extracellular MMP-9 in keratinocytes treated withrecombinant toxin D SMase, (12) reduces disorganization of dermiscollagen fibers, bleeding, inflammatory infiltrate, and injury toadjacent muscle layer in skin contacted with venom, (13) inhibits by92.7% superoxide production by keratinocytes treated with recombinanttoxin D SMase, (14) reverses by 61.1% the production of TNF-α from thesurface of keratinocytes treated with recombinant toxin D SMase, (15)reduces by 89.2% the production of TGF-β1 from the surface ofkeratinocytes treated with recombinant toxin D SMase, (16) reduces by80.2% phosphorylated ERK1/2 in keratinocytes treated with recombinanttoxin D SMase. 15-37. (canceled)
 38. The pharmaceutical composition ofclaim 5, which controls the effects of sphingomyelinases from bacteriaand from arthropods, wherein the arthropods are selected from the groupconsisting of (spiders, scorpions and ticks).
 39. that thepharmaceutical composition of claim 38, wherein the sphingomyelinasesare from bacteria selected from the group consisting ofCoryneumbacterium pseudotuberculosis, Arcanobacterium haemoliticum, andBacillus cereus.